Antianemic substance and process of making it



Patented How s, 1935- I I UNITED STATES PATENT OFF E George B. Walden, Indianapolis, Ind., assignor to Eli Lilly and Company, Indianapolis, Ind, a corporation of No Drawing. Application March 17, 1933, Serial No. 661,356

13 Claims. (Cl. 167-74) It is the object of my invention to obtain a mucous membraneof the stomach. See Thorpes potent anti-anemia substance in concentrated Dictionary of Applied Chemistry, (1913), vol. 4,

form, a p. 112; and Supplemental to American Dispen- It is known (Minot and Murphy, Whipple, and satory, (15th edition, 1886,) by.John King and others) that whole liver and kidney are efiective John U. Lloyd, p. 120. These mere dried stom- 5 in the treatment of anemias generally, both secach-linings, sometimes defatted, which constiondary anemia and pernicious anemia. It is also tuted original commercial pepsin, are now known known (Cohn, Campbell, Walden) that certain to be efiective by oral administration in the treatfractions of these tissues are obtainable that are ment of pernicious anemia. The ordinary pres 19 effective respectively in the treatment of secondant-day commercial pepsin (of the United States.

ary anemia and of pernicious anemia. It is also Pharmacopoeia), however, is not the mere dried known (Sturgis and Isaacs) that whole stomachs lining or mucous membrane of the stomach; but and stomach linings are efiective in the treatis a water-soluble product derived from the mument of anemia, and more'particularly pernicious cous membrane, by fluid-extraction after aut'oly- 10 anemia. sis, to contain in a more concentrated andpuri- Further, it is known (Castle) that beef muscles fied form the proteolytic enzyme, itself also called and stomach secretions are not eflective separatepepsin, which digests egg-white under acid condily in the treatment of pernicious anemia, but that tions and has .little or no activity at neutrality.

a product obtained by mixing beef muscle and It has been shown that such present-day coma 0 stomach secretions and digesting the mixture at mercial pepsin is not only not effective itself to 20 an appropriate hydrogen ion concentration is efrelieve pernicious anemia but cannot be substifective in such treatment, whether such digestion tuted for stomach secretions to co-operate-with is carried on externally of the, patients body on beef muscle to produce a product which is effecan externally made mixture of such muscle and tive in-the treatment of pernicious anemia. (Cassuch stomach secretions, or is carried on within tle, Wilkinsom) a 25 the patients stomach on a mixture resulting upon In my Patent No. 1,894,247, granted January separate feeding of'such muscle and such stom- 10, 1933, I have described a potent anti-anemia ach secretions simultaneously or sufliciently substance consisting of a mixture of certain stomnearly simultaneously so that interaction occurs ach-tissue material with tissue material of the between the muscle and the secretions. These class comprising liver and kidney, preferably dithings are described by Castle in an article in gested extracorporeally to permit the interaction the American Journal of Medical Sciences for Deof the ingredients. cember, 1929. It had earlier been reported by I have also discovered thatit is possible to get v Castle at the meeting of the American Society for a highly effective and highly concentrated acti- Clinical Investigation at Washington on April 30, vated anti-anemia substance by appropriate 1928, that beef muscle ground up andied to a treatment of certain stomach-tissue material normal person, and allowed to digest in the stom alone. That is, using only such stomach-tissue ach ofthat person for a few hours, could then material, I am able to activate it so that I get be removed from the stomach of that person and therefrom an anti-anemia product which is more was effective ,by oral'administration in the treatefiective, often from three tovtwelve times, than ment of pernicious-anemia patients. It was also any single-tissue-derived material previously shown by Castle that stomach-secretions, orally known, compared on the basis of the weight of administered alone, are not eflective in the treatthe original tissue employed; and since, as shown ment of pernicious anemia; and that a treatby the reported work of various investigators, inment is not effective in relieving 'pemicio'us eluding Conner (Jour. A. M. A., February 14, 1931, anemia if stomach secretions and beef muscle pp. 500-503,) and Sharp, Sturgis, and Isaacs are orally administered to the patient at sufii- (U.'S. Patent No. 1,937,133) prior stomach-tissueciently widely separated times to permit the maderived products have been derived from the origterial first so administered to leave thestomach inal stomach tissuefwithout loss of potency, before the second is administered, as for instance the present product is an activated product .which by administration of one early in. the morning has a higher potency than has a weight of origiand of the other inthe afternoon. nal stomach-tissue material equal to that from The use of stomachs as a digestive has also which such activatedproduct was derived, often long been known. For example, the original com-- several fold. mercial pepsin was merely the dried lining or .In this way, I am able toproduce effective re- 2 lief of certain anemias, especially pernicious anemia, by the oral administration of a.da-ily dose of material derived from not to exceed grams, and usually that grams, of the original stomach-tissue material, such as whole stomachs; in contrast to the daily dose necessary, in order to produce comparable results, of from 67 to 300 grams of original stomach tissue, or material derived from at least as much as 6'7 grams of original tissue with any previously known single-tissue-derived anti-anemia product, even the dried and defatted stomachtissue products heretofore used in the treatment of pernicious anemia. The amount actually administered of my material, when in dry form, weighs less than 20%, and usually about 15%, of the original stomach-tissue material from which it is derived, so that my material derived from 40 grams of the original stomach-tissue material weighs about 6 grams, and that derived from 12 to 25 grams of original material weighs from about 1.8 to 3.75 grams; whereas the daily dose which it was necessary to administer of previous singletissue-derived anti-anemia products in order to produce comparable results had a weight of the order of from 10 to grams, and was derived from 67 to 300 grams of original stomach-tissue material.

The stomach-tissue material which I may use should include some or all of the lining of the pouch or central section of the stomach-the longitudinally intermediate portion which includes all of the fundus gland region with varying amounts and often the whole of the cardiac region and of the pyloric region. But the stomach lining alone, even of the pouch or central section, although it has an anti-anemia effectiveness by itself as has been shown by Sturgis and Isaacs and by Conner, does not by my treatment yield a product of the high anti-anemia potency which appropriate stomach-tissue material yields by such treatment. This difference may partly or wholly explain why the ordinary present-day commercial pepsin is substantially ineffective to relieve pernicious anemia, for such present-day commercial pepsin is a water-soluble extract derived by fluid-extraction from stomachlinings alone after they have been subjected to autolysis. My product has an exceptionally high peptic activity-rangingfrom 1:500 to 1:1600 or more-as well as a high potency in relieving pernicious anemia.

Because of the aforesaid situation about stomach lining, it is necessary for my invention to use more of the stomach than the mere lining or a portion of the lining thereof in order to get the high potency which I obtain. This may be done by using the whole stomach-which is a term of considerable inclusiveness, and means generally a whole thickness of stomach wall, and more particularly the entire pouch or central section of the stomach with varying amounts and often the whole of the cardiac and pyloric regions. In any case, in addition to the lining of the pouch or central section of the stomach, stomach-wall tissues other than the mucosa should also be.

used. This combination is what I mean by appropriate stomach-tissue material" as used in this application.

In carrying out my invention, method, I proceed as follows:

1. I use the appropriate stomach-tissue material in fresh condition, as soon as possible after the slaughter of "the animals. I deem it preferable that after slaughter and before processing the in the preferred derived from 12 to 25- stomach-tissue material shall not be allowed to remain for more than five or six hours at a temperature materially higher than freezing; as otherwise the yield of potent material is decreased.

2. I permit the fresh appropriate stomachtissue material, after being washed and conveniently minced or ground, to undergo incipient digestion, so that there may be an interaction between substances present in it. Because the digestion is of that nature, and needs no added 10 ferment or enzyme, I can properly call it selfdigestion; even though, as noted below, I also add acid to obtain the desired hydrogen ion concentration. Thisself-digestion is preferably at a temperature around body temperature (98 F.), 15 although some variation from body temperature is permissible, for a period which may vary over a considerable range but is desirably such that a digestion comparable to normal digestion in the living stomach is obtained. The higher the tem- 20 that the temperature should not be allowed to allowed for the incipient self-digestion. At body temperature, good results are obtained with digestion for from two to twelve hours. While undoubtedly longer periods can be effectively used, 25 I deem it desirable that the total period between the slaughter of the animals and the end of the digestion shall not include more than eighteen (18) hours at which the material is at a temperature substantially above freezing. It is desirable 30 that the temperature should not be allowed to original stomach-tissue exceed 115 F. at any time during or prior to the processing. The self-digestion is incipient in contradistinction to the full or complete digestion or autolysis which liquefies the whole mass, 35 as has been done; for by maintaining the digestion incipient I produce very little if any liquefaction. I do not separate any liquid from the still substantially solid mass, but use the whole mass, save for the removal of water (by evapora- 40 tion) and fats as noted below.

3. This self-digestion is best carried out it cle. acid conditions, desirably between pH 1 an pH 5.5, and conveniently at about pH 4.5 to pH 5. In obtaining the desired hydrogen ion concentra- 45 tion, I deem it desirable to add only acid, and to do so with care not to add too much acid.

4. The incipiently self-digested material is promptly dehydrated, at the end of the self -digestion period, desirably by evaporation in the ab- 50 sence of free access of air and at a temperature continuously kept below 115 F.; and preferably is also defatted. The removal of water and fat, of course, is a substantially universal procedure in the making of gland extracts. This dehydra- 55 tion is most conveniently done under high vacuum, such for instance as under an absolute pressure of a half-inch to one inch of mercury at a temperature not in excess of 115 F.; and I prefer that it be done at an absolute pressure of less so than a half-inch of mercury at a temperature not in excess of F. Subsequent to dehydration, it is desirable to defat, as by extracting the material thoroughly with petroleum ether. I

This produces a solid product, sometimes more as or less powdery but in any case one which can readily be ground to a powder. .It has had nothing removed from the original stomach-tissue material with the exception of water and fat; and its weight is usually about 15% of that of the 70 material from which it was derived. It is heat-labile-not .fully stable when subjected-to heat. Indeed, it is because of this heat-lability, which I think] is mainly a lability of one component which enters into the '18 activating reaction, that the temperature must be kept low during its making. It contains nitrogen. It is substantially insoluble in petroleum ether'and in benzene; and also in acetone, alco-' hol, .and ether. It is partially soluble in water, for about half of it will go into water solution;

and that solution will usually contain about -The product thus obtained, ground if desired,

is found to be very potent in the relief of certain anemia-s, especially pernicious anemia. It is effective in the majority of cases when orally administered in cases of pernicious anemia in daily doses of 6 grams (derived from about 40 grams of original material), and in most cases in daily doses of 128 to 3.75 grams (derived from 12 to 25 grams of original material), in contrast to the daily doses of several times that amount necessary of any single-tissue-derived material, from either stomach alone or liver alone, known prior to my present invention, and in contrast to the daily dose of from 67 (the minimum dose found among prior reports) to 300 grams which has been found necessary of untreated original stomach-tissue material. other liquid, but the weights given for the doses are dry weight, or weights of the solid product.

When administered orally in the doses indicated -of anemia. My extract contains some of these vitamins from the original stomach-tissue material; but because of the small quantity of tissue material. used for a dose, that amount of such 'vitamins is in some cases insuflicientfor best results. In those cases it is desirable that B vitamins in some form he added-to my product. 'I'hislmay be done by-adding them in any of the available forms, such as those derived from wheat embryos, rice polishings, or yeast. The amount of B vitamins so added may be varied as desired;

but good results can be obtained by adding from where on treatment the hemoglobin increases at a lower rate than does,the red-blood-cell count; as iron is an essential constituent of hemoglobin. Some iron, of course, comes from the original tissue, since it is present in all tissues; but this may not be sufficient because of the small quantity of original tissue represented in a dose of my product. The iron salt is desirably in the form I "of iron-ammonium citrate green, although other iron salts may be used.

It may be administered in water or- -one-fifth of the original whole stomachs.

Either the vitamin B or the iron salt may be added alone, or both may be added.

' In making my anti-anemia material, I prefer to use all the steps noted, for best results. However, I do not regard all these steps as essential 5 to my invention in its broad sense. The removal of water and fat is desirable to reduce the bulk and to improve the taste even if the material is to be used immediately, and to prevent the material from becoming rancid in case it is not to be used immediately, as is usually the case. All the steps noted are cumulative in their eifects, to obtain highpotency, stability, and palatability. My process of producing a potent anti-anemia material is susceptible of considerable variation 15 in detail. The following example shows one way of carrying out my invention to produce a highly potent material.

' I Example chloric acid toadjust the hydrogen ion concentration to about pH 4.5 to pH 5. This slightly acid material is thoroughly stirred, and allowed to undergo digestion for 2 to 4 hours at a temperature between 98 and 100 F., so that substances present in the material may interact. Immediately after the desired period of selfdigestion, the incipiently self-digested material is dehydrated, in a vacuum drier, at an absolute pressure of 0.2 to 0.5 inch of mercury and a temperature under 100 F. The dehydrated material is then defatted, as with petroleum ether; and any residual petroleum ether is removed, as by drying.

The dehydrated and defatted material thus 40 obtained from 100 pounds of original whole stomachs usually weighs about 15 pounds-less than This material is a highly eifectiveanti-anemia material embodying my invention, and may be orally 45 administered directly in this form.

If desired, extracts-may be made at any stage in the procedure, as. by expression or water extraction. Such extracts have varying degrees of .potency, although simple water extraction has 50 given only relatively mediocre yields as compared with the unextracted material. These extracts, desirably after being reduced to a powder in vacuo at low temperature, may be administered as such.

Prior products have been made by the removal of water and/or fat from stomach-tissue material, with about the same concentration thereby in bulk and weight from the original stomachtissue material as I get, or to about 15% by weight of the original stomach-tissue material. The v diflerence between my product and these prior products is thus not in the mere matter'of concentration in weight and/or in bulk, or in the greater removalof inert matter. The funda- .5

mental difference is that my process involves a definite activation or potentiation, in the incipient self-digestion or in the acid condition produced, or when both are used as is desirable; as a result 0 of which my product, as distinguished from prior products which at best have suffered no loss in potency, is more potent than a weight of original stomach-tissue material equal to that from which my product was derived. '5

I claim as my invention:

1. A potent anti-anemia product, comprising incipiently self-digested and dehydrated. and defaited whole-stomach. v 2. A potent anti-anemia product, comprising incipiently self-digested and dehydrated and defatted appropriate stomach-tissue material.

3. A potent anti-anemia product, comprising incipiently self-digested and dehydrated and defatted whole-stomach including the fundus gland region and at least substantial portions of the cardiac and pyloric regions.

4. A potent anti-anemia product, comprising an activated substance derived from stomach-tissue material, which product is heat-labile, contains nitrogen, is substantially insoluble in petroleum ether and in benzene, and when orally administered in cases of -pernicious anemia in daily doses of material derived from between 12 and 40 grams of original stomach-tissue material is eiiective in the majority of such cases to produce the recognized symptoms characteristic of the relief of such cases.

5. A potent anti-anemia product, comprising an activated substance derived from stomachtissue material, which product is heat-labile, contains nitrogen, is substantially insoluble in petroleum ether and in benzene, and when orally administered in cases of pernicious anemia in daily doses of material derived from grams of original stomach-tissue material produces results which for their production with original.

stomach-tissue material require doses of from 6'7 to 300 grams.

6. A potent anti-anemia product, comprising an activated substance derived from stomachtissue material, which product is heat-labile, contains nitrogen, is substantially insoluble in petroleum ether and in benzene, and when orally administered in cases of pernicious anemia in daily doses of six grams dry weight is efiective in the majority of such cases to produce the recognized symptoms characteristic of the relief of such cases.

'7. The process of producing a potent antianemia product from appropriate stomach-tissue material, which consists in subjecting such appropriate stomach-tissue material to incipient self-digestion, and dehydrating and defatting the resultant product.

' 8. The process of producing a potent antiv anemia product from appropriate stomach-tissue material, which consists in subjecting such appropriate stomach-tissue material to incipient self-digestion, and drying in the absence of free access of air at a temperature not above 115 F. 5

9. The process of producing a potent antianemia product from whole-stomach, which consists in subjecting such whole-stomach, including the fundus gland region and at least substantial portions of the cardiac and pyloric regions, to 10 incipient self-digestion, and dehydrating and defatting the resultant product.

- 10. The process of producing a potent antianemia product from stomach-tissue material, which consists in subjecting the stomach-tissue 1| material to incipient self-digestion under acid. conditions at a temperature which does not exceed 115 F. and in the substantial absence of other tissue, and dehydrating.

11. An activated product derived from whole- M stomach tissue; which product is heat-labile,. contains nitrogen, is substantially insoluble in petroleum ether and benzene, has a peptic activity in excess of 1:500, and in daily oral doses of six grams dry weight is effective in the majority 86 of pernicious-anemia cases to produce the recognized symptoms characteristic of the relief of such cases.

12. An activated product derived from appropriate stomach-tissue material; which product '0 is heat-labile, contains nitrogen, is substantially insoluble in petroleum ether and benzene, has a peptic activity in excess of 1:500, and in daily oral doses of six grams dry weight is effective in the majority of pernicious-anemia cases to pro- 8 duce the recognized symptoms characteristic of the relief of such cases.

13. The process of producing a potent antianemia product from a stomach-tissue material,

which consists'in adding acid to such stomach- 40 tissue material to produce a hydrogen ion concentration of between pH 1 and pH 5.5, and causing self-digestion to start by holding such sotreated stomach-tissue material fora period at a temperature which is conducive to selt-digestion and which does not exceed F. but dehydrating such stomach-tissue material before it has been allowed after slaughter to remain at a temperature materially above 98 F. for more than twelve hours.

CERTIFICATE OF CORRECTION.

Patent N 2, 020, 267 d November s, 1935.

GEORGE B. .WALDEN.

it is hereby certified "that error appears in theprintedspecificatibn ol the a above. numbered patent requiring correction as follows: Page 1, second column,

I lme 3, forVSupplemehtai" rend-Supplement; line 32, after "ingredients" and before the period insert the words upon each other; page 2, second column, strike out line 21, and insert instead the syllable and words perature ,the shorter theperoid. which should be; and that the said Letters Patent should be. read wilh this correction therein that the same may conform to the record of the .case in the ,Patent Office.

Signed and sealedthis 17m one-amber, o. 1935.

Les! ie' Frazer (Seal) Q Acting Commissioner of Patents. 

